Biology Reference
In-Depth Information
Table 2
Biochemical methods established for purity assessment of Arabidopsis leaf peroxisomes
Enzymatic assay
Immunoblotting
Organelle
Marker enzyme
Reference
Marker protein
Reference
Leaf peroxisomes HPR
[
7
,
8
,
20
]
APX
[
21
]
Catalase
[
9
,
10
,
22
-
24
] Catalase
[
9
,
10
,
21
]
KAT2
[
10
]
Mitochondria
Fumarase
[
7
,
20
,
23
,
24
] VDAC
[
8
]
Cytochrome C oxidase
[
9
,
21
]
NADH dehydrogenase [
22
]
Cpn10
[
9
]
Chloroplasts
NADP dependent GAPDH [
7
,
20
]
PSI-D (thylakoids)
[
25
]
Cpn20
[
9
]
ER
NADH:Cyt
c
reductase
[
23
]
BiP
[
9
,
22
]
Calnexin [
22
]
The following acronyms were used:
APX
ascorbate peroxidase,
BiP
binding protein,
Cpn10/20
chaperonin 10/20,
HPR
hydroxypyruvate reductase,
KAT2 thiolase
,
NADP-GAPDH
NADP dependent glyceraldehyde dehydrogenase,
PSI-D
photosystem I subunit D,
SSU
small subunit of RubisCO,
VDAC
30-kD voltage-dependent anion-selective
channel
5. Filter the ground tissue through miracloth (1-3 layers) into an
Erlenmeyer fl ask to obtain the crude extract (CE). Gently
squeeze the miracloth to collect the entire CE in the fl ask. If
the CE is to be analyzed (e.g., for marker enzyme activities
such as hydroxypyruvate reductase, HPR, for leaf peroxisomes,
see
Table
2
), aliquots are taken (
see
Notes 12
and
13
).
6. Pour the CE about equally into six SS34 tubes (polypropylene
or polycarbonate), balance and centrifuge them for 1 min at
5000
× g
(Sorvall SS34) to sediment chloroplasts and nuclei
(
see
Note 14
).
7. Pour the six supernatants into a precooled 300-mL beaker
(
see
Note 15
). Layer carefully about 19 mL on top of each of
the eight SS34 Percoll gradients (
see
item 9
of Subheading
2.2
).
8. Centrifuge the Percoll gradients for 12 min at 13,000 ×
g
at 4 °C
in a Sorvall SS34 rotor and then increase the speed to 27,000 ×
g
and spin for another 20 min (no slow start, brake on).
9. Carefully suck off the top fractions of the gradients using a
short Pasteur glass pipette attached to a vacuum pump. Take
off the entire green 15/38 % (v/v) Percoll interface (chloro-
plasts/thylakoid fraction, Fig.
1
) while removing only a minor
